Knowledge and Management of Aquatic Ecosystems (2014) 414, 12 http://www.kmae-journal.org c ONEMA, 2014 DOI: 10.1051/kmae/2014024

Infestation of cyprinacea (Copepoda: ) in two invasive fish species in Romania, Lepomis gibbosus and Pseudorasbora parva

M.M. Stavrescu-Bedivan(1),O.P.Popa(2),L.O.Popa(2),

Received May 14, 2014 Revised July 14, 2014 Accepted July 17, 2014

ABSTRACT

Key-words: In this study we analyzed comparatively the host-parasite associations non-indigenous between two fish host species invasive in Europe (Lepomis gibbosus and fish, Pseudorasbora parva) and one known generalist parasite species, the host-parasite . We used a fragment of the hypervariable coevolution, region D1-D2 of the 28S rRNA to confirm that the copepod specimens quantitative collected on both host species in our study are indeed conspecific. The parasitology, prevalence of infection was significantly different between the two host DNA barcoding, species in all three aquatic ecosystems. Two populations of L. gibbosus molecular exhibited a positive correlation coefficient between the standard body markers length and infection intensity, while a negative correlation coefficient was observed in one population of P. par v a. This is one of the few studies pro- viding parasitological parameters of infections of Lernaea cypriancea in Lepomis gibbosus and Pseudorasbora parva.

RÉSUMÉ

Infestation de Lernaea cyprinacea (Copépode : Lernaeidae) sur des espèces envahis- santes de poissons Lepomis gibbosus et Pseudorasbora parva en Roumanie

Mots-clés : Dans cette étude, nous avons analysé comparativement les associations hôte- poissons parasite entre deux espèces hôtes de poissons envahissantes en Europe (Lepomis non indigènes, gibbosus et Pseudorasbora parva) et une espèce de parasite généraliste connue, coévolution le copépode Lernaea cyprinacea. Nous avons utilisé un fragment de la région hy- hôte-parasite, pervariable D1-D2 de l’ARNr 28S pour confirmer que les échantillons de copé- parasitologie podes prélevés sur les deux espèces hôtes dans notre étude sont en effet de la quantitative, même espèce. La prévalence de l’infection était significativement différente entre ADN barcoding, les deux espèces hôtes dans les trois écosystèmes aquatiques. Deux populations de L. gibbosus présentaient un coefficient de corrélation positif entre la longueur marqueurs standard du corps et l’intensité de l’infection, tandis qu’un coefficient de corréla- moléculaires tion négatif a été observé dans une population de P. p a r v a . C’est l’une des rares études fournissant des paramètres parasitologiques de l’infection de Lernaea cy- priancea sur Lepomis gibbosus et Pseudorasbora parva.

(1) Faculty of Agriculture, University of Agronomic Sciences and Veterinary Medicine of Bucharest, Bucharest, Romania (2) Molecular Biology Department, “Grigore Antipa” National Museum of Natural History, 011341 Bucharest, Romania  Corresponding author: [email protected]

Article published by EDP Sciences M.M. Stavrescu-Bedivan et al.: Knowl. Managt. Aquatic Ecosyst. (2014) 414, 12

INTRODUCTION

Host-parasite coevolution may lead to either host or parasite local adaptation in spatiotem- porally varying environments, e.g. when invasive species expand their range (Lemoine et al., 2012). One of the factors considered to explain the success of these species is the release of the newly founded populations from the pressure exercised by their original parasites left be- hind in the initial environment (Mastitsky et al., 2014;Torchinet al., 2003). This theory relies on the idea that co-evolution leads to extensive local adaptations, and as such the parasites in a particular area infect hosts from the same area more efficiently than they infect hosts from an- other geographically distinct population (Morand et al., 1996;Rothet al., 2012). On the other hand, since the newly conquered environments are not parasite-free, the new host-parasite interactions can lead to parasite maladaptation, where parasites can temporarily have higher fitness in the newly arrived (allopatric) hosts than in the sympatric hosts (Dybdahl and Storfer, 2003). In this study we analyzed comparatively the host-parasite associations between two fish host species invasive in Europe (Lepomis gibbosus and Pseudorasbora parva) and one known generalist parasite species, the copepod Lernaea cyprinacea. Lepomis gibbosus (Linnaeus, 1758) is a freshwater fish species native to North America, be- longing to the family Centrarchidae. Introductions of pumpkinseed sunfish beyond its native range have occurred around the world for many years, and for many reasons, such as poten- tial sport, ornamental pond fish, fry and fingerlings, and prey for native wild stocks (Jordan, 2009). Introduced to Europe in the 1880s as a pond and aquarian fish, the species is quite widespread throughout this continent, being particularly abundant in Mediterranean coun- tries (Hanel et al., 2011). The second host in our study, Pseudorasbora parva (Temminck & Schlegel, 1846), is an Asian native freshwater cyprinid species considered a typical invasive species and occurring on all continents except for Antarctica (Margaritov and Kiritsis, 2011). The accidental transfer and release of P. par v a with carp species translocations for aquacul- ture is considered the primary pathway of species introduction into new areas (Gozlan, 2012). After the first introduction into Europe in the 1960s (Nucet, Romania), the species extended its range through natural dispersal, recreational fishing and ornamental fish trade. Its high pheno- typic plasticity and the ability to cope with novel pathogens have predisposed the topmouth gudgeon to being a strong invader (Hanel et al., 2011). The parasite species, the anchor worm Lernaea cyprinacea Linnaeus, 1758, is the only cosmopolitan species in the genus Lernaea (Piasecki et al., 2004). This copepod is non-specific, infecting many freshwater fish species (Williams and Bunkley-Williams, 1996), but also affecting marine fish hosts (Gutierrez-Galindo and Lacasa-Millan, 2005), as well as adult and larval amphibians (Kupferberg et al., 2009; Nagasawa et al., 2007) and larval aquatic insects (McAllister et al., 2011). L. cyprinacea is native to Asia, but currently it is widely distributed (Oscoz, 2010). The aim of the present study was to compare the parasitological parameters of L. cyprinacea infections on the two invasive fish species in several lentic systems in Romania.

MATERIALS AND METHODS

We conducted parasite surveys of fish fauna during the 2008 summer season in three Ro- manian lentic ecosystems: Moara Domneasca Lake, Ilfov County – 46◦4356N, 27◦4441E (2 surveys in June and July); Brates ¸ Lake, Gala¸ti County – 45◦2859N, 28◦413E(1survey in July); Crapina Lake, Tulcea county – 45◦2224N, 28◦1442E (1 survey in July). The fish specimens were collected by electrofishing with a Samus 720 MP device (Samus Special Electronics, Poland) and immediately after the sampling, they were placed in 95% ethanol and transported to the laboratory. Further analyses of parasite fauna were performed only on L. gibbosus and P. par v a, because both species exhibited a consistent abundance in all samples. For each fish specimen, the standardized length was measured with a calliper to the nearest 0.1 mm. Each fish specimen was examined using a Krüss Optronic dissecting microscope at the level of the branchial cavities, head, fins and skin for presence of copepod

12p2 M.M. Stavrescu-Bedivan et al.: Knowl. Managt. Aquatic Ecosyst. (2014) 414, 12 parasites. The copepod specimens removed were placed in 95% ethanol for further analysis. The morphological identification of the L. cyprinacea specimens was performed according to Bauer (1987). The copepod preference for a particular attachment site on the host was recorded and 95% confidence intervals of the parasite proportions on different host regions were computed with the modified Wald method (Agresti and Coull, 1998). The infestation parameters prevalence, mean intensity, median intensity and mean abundance, as well as the corresponding 95% C.I., were calculated using the software package Quantitative Para- sitology 3.0 (Rozsa et al., 2000). The Spearman rank correlation coefficient (rs) was used to measure correlation between host fish size and the abundance of infection, while a p-value for the null hypothesis of rs being equal to zero was computed based on 10000 Monte Carlo replications using the same software package. The comparison of prevalence between popu- lations of the same host species and between host species in the same lake were performed by Fisher’s exact test. In all cases p values lower than 0.05 were considered significant. We tested that the copepod individuals collected on both fish hosts are conspecific (L. cypri- nacea) by DNA sequencing of a fragment of the 28S rDNA. Total DNA was extracted from eight copepod individuals, from both host species, using the NucleoSpinR Tissue kit (Macherey–Nagel GmbH & Co. KG, Düren, Germany), according to the manufacturer’s specifications. The amplification of a fragment of the hypervariable region D1-D2 of the 28S rDNA fragment was performed with the primers 28SF (5–acaactgtgatgcccttag–3) and 28SR (5–tggtccgtgtttcaagacg–3)(Songet al., 2008). The amplification was performed in a 50 µL reaction containing 10 mM Tris–HCl (pH 8.8 at 25 ◦C), 50 mM KCl, 0.08% (v/v) Nonidet P40, 2.5 mM MgCl2, each dNTP at 0.1 mM, each primer at 0.2 µM, 1 unit of Taq DNA polymerase (Fermentas UAB, Vilnius, Lithuania), and approximately 50 ng of DNA template. The temper- ature profile of the polymerase chain reaction for the 28S marker consisted of initial denatu- ration at 94 ◦C for 30s, followed by 30 cycles at 93 ◦C for 30s, 50 ◦C for 45s, 72 ◦C for 45s, and a final extension step performed at 72 ◦C for 10 min. The PCR products were separated on 1% agarose gel, and the fragments of interest purified using the innuPREP DOUBLEpure Kit (Analytik Jena AG, Jena, Germany). The DNA sequencing was performed by Macrogen Europe (1105 AZ, Amsterdam).

RESULTS

During the 2008 summer surveys 312 specimens of Lepomis gibbosus and 393 specimens of Pseudorasbora parva were collected. A total of 224 Lernaea cyprinacea adult females were collected from both Lepomis gibbosus (199) and Pseudorasbora parva (25) fish hosts, with a range of 1 to 11 parasite specimens per host. The sample sizes, mean body measure- ments, copepod infestation parameters and correlation coefficients between the host stan- dard body length and abundance of infection are displayed in Table I, while the comparison of prevalence between populations/species is presented in Table II. The attaching preference of L. cyprinacea for a particular host body area in L. gibbosus is recorded in Table III.In our study two populations of L. gibbosus exhibited a positive correlation coefficient between the standard body length and infection intensity, while a negative correlation coefficient was observed in one population of P. par v a. The comparison of prevalence (Table II) revealed sig- nificant differences between the L. gibbosus sample collected in Moara Domneasca in June 2008 and all the other L. gibbosus samples. On the other hand, all three L. gibbosus vs. P. par v a comparisons revealed significant differences concerning the prevalence, while no significant differences were observed among the P. par v a samples. The parasite preference for host attachment in L. gibbosus shows that in the Moara Domneasca samples, the skin and head were the preferred attachment areas over the fins, while the opposite situation could be observed in the remaining two fish samples (Table III). A 417bp fragment of the hypervariable D1-D2 region of the rRNA 28S was successfully se- quenced from 8 specimens of Lernaea sp. from both host species. Only one haplotype was identified in all cases (GenBank accession numbers KF751648.2- KF751649.2). A BLAST

12p3 M.M. Stavrescu-Bedivan et al.: Knowl. Managt. Aquatic Ecosyst. (2014) 414, 12 c s confidence r 0.13 0.07 0.38 0.25 –0.15 –0.08 –0.21 -value) (0.0025) (0.2573) (0.6110) (0.0289) (0.3972) (0.0193) (0.5201) p ( b 0.5 0.975 0.061 0.066 0.326 0.586 0.059 Mean (0.206–1) (0–0.176) (95% C.I.) abundance (0.725–1.25) (0.428–0.842) (0.174–0.478) (0.020–0.135) (0.035–0.097) values significantly different from zero. b s – 1 1 1 1 1 1 (1–2) (1–3) (1–3) pper-Pearson confidence interval; b – Median intensity (95% C.I.) b 1 1 1.66 1.89 1.15 1.29 1.71 Mean (1–1.31) (1.22–3) (1–1.86) intensity (1.38–2.46) (1.43–2.02) (95% C.I.) a 0.588 0.265 0.066 0.283 0.047 0.342 0.059 (95% C.I.) Prevalence rs. SBL – standard body length; a – Clo (0.267–0.423) (0.160–0.435) (0.472–0.696) (0.129–0.444) (0.037–0.106) (0.019–0.095) (0.001–0.287) 74.7 65.6 36.7 60.7 38.6 57.6 49.2 (40–97) (17–80) (47–85) (20–61) (20–78) (47–76) (45–101) Mean SBL (min–max) 80 34 46 17 152 228 148 size Sample Host P. p a r v a P. p a r v a P. p a r v a L. gibbosus L. gibbosus L. gibbosus L. gibbosus s Lake Sampling site Moara Domneasca Lake June 2008 Moara Domneasca Lake July 2008 Brate ¸ July 2008 Crapina Lake July 2008 Table I The sample sizes, mean body measurements and copepod infestation paramete interval computed with 2000 bootstrap replications; c – p-value based on 10000 Monte Carlo replications; gray cells indicate r

12p4 M.M. Stavrescu-Bedivan et al.: Knowl. Managt. Aquatic Ecosyst. (2014) 414, 12

Table II Two-sided p-values for comparison of prevalence by Fisher’s exact test; gray cells indicate significant differences. L. gibbosus P. p a r v a MD_Jul Brates ¸ Crapina MD_ Jul Brates ¸ Crapina MD_Jun 0.0020 0.0015 0.0005 MD_Jul 1.0000 0.4261 0.0012 Brates ¸ 0.4800 <0.0001 Crapina 0.0241 L. gibbosus MD_Jul 0.5081 1 Brates ¸ 0.5893 P. p a r v a query run against the Genbank Nucleotide collection (nr/nt) returned a species match with Lernaea cyprinacea at similarity levels between 100% and 99.76% (3 matches). The next best match scored a 91% similarity with Lamproglena orientalis Markevich, 1936.

DISCUSSIONS > L. CYPRINACEA SPECIES CONFIRMATION The origin and taxonomic position of Lernaea cyprinacea is open to discussion. According to (Margaritov and Kiritsis, 2011), L. cyprinacea is a European parasite, whereas L. elegans is considered to be an Asian species. However, the majority of experts regard both species as synonyms (Nagasawa et al., 2007). Given this controversy, we used a fragment of the hy- pervariable region D1-D2 of the 28S rRNA to confirm that the copepod specimens collected on both host species in our study are indeed conspecific. The hypervariable regions of 28S rRNA have been used by other authors as species diagnostic markers in , annelids and mollusks (Markmann and Tautz, 2005; Raupach et al., 2010;Songet al., 2008). All eight examined sequences (from two host species) were identical. Moreover, the BLAST query run against the Genbank Nucleotide collection (nr/nt) returned a species match with L. cypri- nacea. These results corroborated with the morphological identification performed according to Bauer (1987) confirmed that in our study we analyzed L. cyprinacea specimens on two different host species.

> PREVIOUS RECORDS OF THE PARASITE AND/OR THE HOST SPECIES

Recorded in Romania for the first time in 1929 by Busnita, the pumpkinseed sunfish L. gib- bosus is currently present in almost all limnic ecosystems in the country (Gavriloaie et al., 2006). Banarescu (1946) found for the first time the anchor worm L. cyprinacea parasitizing L. gibbosus in Romania and the same author provided a detailed list of the pumpkinseed sunfish parasites in 1964 (Banarescu, 1964). More recently, Stavrescu-Bedivan et al. (2011) reported the anchor worm from L. gibbosus. Radulescu and Georgescu (1969)studiedfor thefirsttimetheparasitesofP. par v a in Romanian waters, identifying one protozoan and one nematode species, while Banarescu (1999) cited three protozoan species. Stavrescu-Bedivan et al. (2012) reported for the first time in Romania the presence of L. cyprinacea as a parasite on P. par v a. Lernaea cyprinacea was also reported in Romania from the fish species Caras- sius auratus, Cyprinus carpio, Rhodeus sericeus amarus and Rutilus rutilus (Cojocaru, 2003; Patriche et al., 2009).

> DIFFERENCES IN PARASITOLOGICAL PARAMETERS

In our study, the prevalence of infection was significantly different between the two host species in all three aquatic ecosystems. This finding could be interpreted in different ways,

12p5 M.M. Stavrescu-Bedivan et al.: Knowl. Managt. Aquatic Ecosyst. (2014) 414, 12 ; 2 0 0 0 n % 2.2 0.0 0.0 0.0 0–4.0 0–16.2 0–18.0 0.1–8.3 95%C.I. Nasal cavity 0 1 0 0 n % 0.0 0.0 1.3 0.0 Eyes 0–7.6 0–3.6 0–16.2 0–18.0 95%C.I. 4 9 2 1 n % 4.5 6.7 11.5 11.8 0–31.8 95%C.I. 6.0–20.7 1.4–11.4 2.0–35.6 Oral cavity 4 1 n % 12 20 6.7 13.5 25.6 23.5 0–31.84 95%C.I. 9.1–47.8 7.73–22.3 17.19–36.38 Opercular area 0 4 1 0 n % 0.0 0.0 5.9 5.1 Gills 0–3.6 0–18.0 0–28.9 95%C.I. 1.6–12.9 3 0 1 1 n C % 3.4 6.7 0.0 5.9 ns; P – pectoral fins; V – ventral fins; D – dorsal fin; A – anal fin; C – caudal fin 0–4.0 0–31.9 0–28.9 0.7–9.9 95%C.I. 8 7 1 1 n A % 9.0 6.7 9.0 5.9 0–31.9 0–28.9 95%C.I. 4.4–17.0 4.2–17.7 9 0 6 n D % 20 0.0 22.5 40.0 11.5 0–16.2 95%C.I. 6.0–20.7 15.0–32.4 19.8–64.3 5 1 4 V n % 15 5.9 6.4 16.9 26.7 0–28.9 95%C.I. 2.4–14.5 10.4–26.1 10.5–52.4 5 5 1 n P % 19 6.7 6.4 21.3 29.4 0–31.8 95%C.I. 2.4–14.5 13.0–53.4 14.0–31.0 2 0 6 n % 18 6.7 0.0 23.1 11.8 Skin 0–18.0 95%C.I. 2.0–35.6 2.9–14.2 15.0–33.6 s L. gibbosus MD Jun. 2008 L. gibbosus MD Jul. 2008 L. gibbosus Brate ¸ Jul. 2008 L. gibbosus Crapina Jul. 2008 Table III Parasite attachment preference on L. gibbosus. (n) – number of copepod specime MD – Moara Domneasca; light gray cells-fins; dark gray cells-head.

12p6 M.M. Stavrescu-Bedivan et al.: Knowl. Managt. Aquatic Ecosyst. (2014) 414, 12 considering the geographic origin of the species involved. Firstly, we have an Asian parasitic species, one Asian host (P. par v a) and one North-American host (L. gibbosus). Although our data fits the rare model of the sympatric host having developed efficient defense mecha- nisms against its sympatric parasite (Morand et al., 1996), the data we have are not enough to support this hypothesis. We believe the more likely explanation in this case is the gen- eral resistance of P. par v a to various parasite infections observed by some authors (Hanel et al., 2011). In fact, similar low levels of prevalence and intensity of L. cyprinacea infection in P. par v a to those in our study have been reported in the literature (Margaritov and Kiritsis, 2011). The above-mentioned parasite resistance actually refers to the resistance to the pos- sible damaging effects of the parasite, the topmouth gudgeon being otherwise known as a healthy carrier for different parasite species (Spikmans et al., 2013) or being a host for Unonid glochidia, a relation which some authors define more as commensalism (phoresy) than para- sitism (Douda et al., 2012). Secondly, we have a host-parasite combination, L. gibbosus and L. cyprinacea, which have lived in sympatry in Europe for more than 100 years, and a newly arrived host species population (P. par v a). Looking at our data this way, it fits the general model of a parasite species being more effective at infecting the sympatric rather than the allopatric host, showing that the parasite has had more time to develop adaptations for in- fecting the sympatric host species (Lemoine et al., 2012). However, the differences observed in the parasitological parameters between our host species could also be explained by eco- logical factors (local conditions, proximity of host and parasite in time and space, etc.), not only by co-evolutionary driven physiological compatibility, as discussed above (Poulin, 2006). In fact, the results discussed in the next paragraph suggest that the ecological conditions could be an important factor explaining our data. Besides the difference in prevalence between the two host species, our data showed signif- icant differences in the infection prevalence in L. gibbosus within the same aquatic system, between two consecutive surveys (June and July 2008). These results are probably explained by the temporal variability of parasite outbreaks. It is known that infestations with L. cypri- nacea are more prevalent in the summer months, with water temperatures ranging from 25 to 28 ◦C, when this parasite finds excellent conditions for reproduction (Raissy et al., 2013). An- other possible explanation of these results refers to microhabitat differences between species resulting in different probabilities of the parasite infection.

> HOST SITE ATTACHMENT PREFERENCE

Although L. cyprinacea seems to have a random choice for attachment on the body host, we noted that the parasites were fixed especially on the fins and skin. These observations were previously reported in the literature (Barzegar and Jalali, 2009; Jalali et al., 2008;Kimet al., 2002). One of the hypotheses proposed to explain this copepod attachment preference is that fins offer greater protection against currents and tissues at the base of the fins may be more easily penetrated (Gutierrez-Galindo and Lacasa-Millan, 2005; Iqbal et al., 2012).

> HOST SIZE-LEVEL OF INFESTATION CORRELATION

Two of the studied L. gibbosus populations exhibited a significant positive correlation between the host size and the intensity of infection, results which are in agreement with the studies conducted by Gutierrez-Galindo and Lacasa-Millan (2005)andPérez-Bote(2000). On the other hand, one of the P. par v a populations exhibited a negative correlation coefficient, which may be due to the development of acquired immunity in older fish specimens (Kanwal et al., 2012; Tasawar et al., 2009). This is one of the few studies providing parasitic parameters of infections of Lernaea cypri- nacea in Lepomis gibbosus and Pseudorasbora parva in an environment where both host species are invasive. We found significant differences in prevalence and intensity between

12p7 M.M. Stavrescu-Bedivan et al.: Knowl. Managt. Aquatic Ecosyst. (2014) 414, 12 the two host species, but further studies are necessary to determine if the observed differ- ences are generated by host-parasite coevolution in new environments, by one of the species (P. par v a) exhibiting a general resistance to parasite infections, by extrinsic factors affecting both the host and the parasite populations, or maybe by a combination of any of the above- mentioned factors.

ACKNOWLEDGEMENTS

We would like to thank Prof. Dr. Viorica Balan, the director of the PN II Research Program, Contract SAFAR 051-088/2007, from which this study was partially funded. Oana Popa and Luis Popa were supported by the Grant PD Human Resources no. 36/2013 from the National University Research Council allotted to Oana Popa. We thank Costica Adam and Elena Iorgu for their valuable comments on the manuscript.

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